The blood comprises lipoproteins such as high density lipoprotein (hereinafter abbreviated as HDL), low density lipoprotein (hereinafter abbreviated as LDL), very low density lipoprotein (hereinafter abbreviated as VLDL), and chylomicron (hereinafter abbreviated as CM). These lipoproteins are different in the percentage of components such as cholesterol, triglyceride, phospholipid, and protein, and have different functions in vivo. A lipoprotein mainly comprises 3 types of phospholipids, i.e., phosphatidylcholine (hereinafter abbreviated as PC), lysophosphatidylcholine (hereinafter abbreviated as LPC), and sphingomyelin, thereinafter abbreviated as SM).
PC and SM are major phospholipids among these 3 types of phospholipids and account for approximately 70% and 20% of the total phospholipids, respectively. SM is known to accumulate in atheroma in human and animal models. LDL present in human arteriosclerotic lesions comprises a large amount of SM compared to LDL in the plasma (Non-patent Document 1).
Clinical researches in humans also showed that both of plasma SM and SM/PC ratio are independent risk factors for ischemic heart disease (Non-patent Document 2 to 4).
Hitherto, a method using thin-layer chromatography and a method using high-performance liquid chromatography were reported as a method for measuring SM (Non-patent Document 5); however, they have drawbacks such as being cumbersome in operation and requiring a long time for measurement. An enzymatic measurement method utilizing bacterial sphingomyelinase was also reported (Patent Document 1 and Non-patent Document 2). This measurement method is a method for measuring sphingomyelin by hydrolyzing sphingomyelin to phosphorylcholine and n-acylsphingosine by bacterial sphingomyelinase, hydrolyzing the formed phosphorylcholine to choline by alkaline phosphatase, reacting the formed choline with choline oxidase, and measuring the formed hydrogen peroxide. However, the measurement method has problems, such as influence of the use of alkaline phosphatase on the measurement of other components to be measured and specificity of sphingomyelinase of reacting with LPC as well as SM (Non-patent Document 6).